Nanopore-based single DNA molecule characterization, identification and isolation using speed bumps

ABSTRACT

The present invention relates to a method of using nanopores to obtain sequence information of sample DNAs in ss test DNAs. The method comprises using speed bumps to stall the ss test DNAs in the nanopores at random positions of the ss test DNAs to obtain sequence information of each and every nucleotides of the sample DNAs, and to construct the whole sequences of the sample DNAs. The present invention also relates to identification and/or isolation of test DNAs having desired sequence(s) using nanopore detectors facilitated by speed bump.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/386,888, filed Apr. 17, 2019, which is a continuation ofU.S. patent application Ser. No. 15/435,206, filed Feb. 16, 2017, nowU.S. Pat. No. 10,400,278, which is a continuation of U.S. patentapplication Ser. No. 14/471,051, filed Aug. 28, 2014, now U.S. Pat. No.9,617,593, which is a continuation of U.S. patent application Ser. No.13/333,932, filed Dec. 21, 2011, now U.S. Pat. No. 8,845,880, whichclaims priority to U.S. Provisional Application Nos. 61/426,322, filedDec. 22, 2010 and 61/426,323, filed Dec. 22, 2010; each of which areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 25, 2021, isnamed 04338-004US6_ST25.txt and is 2 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates to a method of DNA sequencing andisolation using nanopore detectors in the presence of speed bumps.

BACKGROUND OF THE INVENTION

A nanopore is a nanometer-scale pore providing the sole pathway for anionic current. An exemplary nanopore can be created from depositingalpha homolysin onto the surface of a lipid bilayer under electricalstimulus, as described in US Application Publication No. 2011/0193570,which are herein incorporated by reference in their entireties.

Nanopore sequencing has the potential to become a direct, fast, andinexpensive DNA sequencing technology. Ideally, individual nucleotidesof a single-stranded (ss) DNA passing through a nanopore will uniquelymodulate an ionic current flowing through the nanopore, allowing therecord of the current to provide DNA sequence information. However, acommon challenge to nanopore sequencing is that the ss test DNAtranslocation is rapid, and the electrical signals obtained cannot beresolved for reliable DNA sequencing. DNA duplex sections have been usedto slow translocation of a ss test DNA to provide more resolvableelectrical signals. However, as the currently available methods conductnanopore detection at about room temperature or higher, a relativelylong DNA duplex section is required to stall the ss test DNA in thenanopore.

Thus, there is a need to provide a method to sequence an unknown DNAusing nanopore technology.

SUMMARY OF THE INVENTION

One aspect of the invention relates to characterization andidentification of a sample DNA using a nanopore detector facilitated bya random speed bump pool.

Another aspect of the invention relates to characterization andidentification of multiple sample DNAs using multiple nanopore detectorsfacilitated by speed bumps.

Another aspect of the invention relates to characterization,identification and isolation of desired sample DNAs using multiplenanopore detectors facilitated by speed bumps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an example of a nanopore detector showing: [A]Electrodes; [B1] Treated Hydrophobic/Lipophilic Semiconductor Surface;[B2] Semiconductor substrate containing electronic circuitry; [C]Solvent/Lipid; [D] Lipid Bilayer; [E] Pore; [F] Conductive solution(salt solution); [G] Chip Packaging; and, [H] Peltier device forelectronic temperature control.

FIG. 2 illustrates the passage of a single stranded (ss) test DNAmolecule through a nanopore.

FIG. 3 illustrates a bulky structure formed at the trailing end of a sstest DNA molecule to stall the passage of the ss test DNA through ananopore.

FIG. 4 illustrate multiple speed bumps bound to a ss test DNA molecule,wherein the ss test DNA is trapped in a nanopore by having bulkystructures on both ends.

FIG. 5 illustrates different binding patterns achieved by contacting ass test DNA with a random speed bump pool.

FIG. 6 illustrates different sequence information patterns achieved byrandomly stalling a ss test DNA in a nanopore to obtain sequenceinformation.

FIG. 7 illustrates a speed bump bound to a ss test DNA having a bulkystructure at a first end to stall its passage through a nanopore.

FIG. 8 illustrates multiple sets of electrical signals obtained by ananopore detector according to the present invention.

FIG. 9 illustrates detection of direction identifier in a ss test DNAtrapped in a nanopore bound by two bulky structures

FIG. 10 illustrates detection of an identifier by an identifier-specificspeed bump.

FIG. 11 illustrates an example of a ss test DNA comprising a sample DNAand multiple functional moieties.

FIG. 12 illustrates an example of a ds test DNA comprising a sample DNAand multiple functional moieties.

FIG. 13 illustrates a ss test DNA trapped in a nanopore bound withmultiple speed bumps on both sides of the nanopore.

FIG. 14 illustrates contacting a ss test DNA with a speed bump train.

FIG. 15 illustrates a flowchart of a process according to one embodimentof the present disclosure.

FIG. 16 illustrates the relationship between working temperature andcapture of a ss test DNA having BS2-1 on one end and a BS1 on the otherend in a nanopore.

FIG. 17 illustrates a melting curve of an exemplary bulky structure(BS2-2).

FIG. 18 illustrates the relationship between electric potential appliedand dwelling time of speed hump-test DNA duplex in a nanopore.

DETAILED DESCRIPTION OF THE INVENTION Definitions

A sample DNA comprises a DNA of interest, which can be a single-strandedsample DNA (ss sample DNA) or a double-stranded sample DNA (ds sampleDNA). The sample DNA can be a natural. DNA obtained from a biologicalsample or a synthetic DNA. The synthetic DNA may be a DNA obtained bymodification of a natural DNA, such as pre-processed. DNA intended foruse in DNA identification and/or sequencing. Examples of suchpre-processings include, without limitation, enrichment of the sampleDNA for desired fragments, paired-end processing, mated pair readprocessing, epigenetic pre-processing including bisulfide treatment,focused fragment analysis via PCR, PCR fragment sequencing, and shortDNA fragment analysis.

A test DNA, as used herein, is a DNA molecule that passes through ananopore for detection purposes. A test DNA can be a single-strandedtest DNA (ss test DNA) and a double-stranded test DNA (ds test DNA). Ass test DNA, as used herein, comprises a section of ss DNA that is to bebound by a speed bump in a method described herein. A ss test DNA mayfurther comprise a sample DNA and other functional moieties (e.g.pre-bulky structure, identifiers and isolation tags).

A pre-bulky structure, as used herein, is an oligonucleotide structurein a DNA molecule which can form a bulky structure under certainconditions. The pre-bulky structure can be a ss DNA or a ds DNA.

A bulky structure, as used herein, is nucleotide structure formed from apre-bulky structure in a ss test DNA molecule. The bulky structurestalls the test DNA molecule in a nanopore at a working condition untilthe working condition is changed to another condition wherein the bulkystructure is converted to the pre-bulky structure or other structuresthat cannot stall the test DNA molecule any more. Examples of bulkystructures include, without limitation, 2-D and 3-D structures such asDNA duplex structures, DNA hairpin structures, multi-hairpin structuresand multi-arm structures.

A nanopore, as used herein, is a pore of nanometer dimensions formed bya pore-forming protein being inserted into a membrane. Typically, themembrane is an electrically insulating lipid bilayer membrane.Pore-forming proteins, such as alpha hemolysin and MspA porin, areinserted into the membrane to form nanopores through the proteinmolecule and the membrane. Electrophysiology measurements can be takenby measuring the ionic current passing through a nanopore as a voltageis applied across the membrane. A nanopore can be utilized as a moleculedetector by monitoring the ionic current variation in response to thepassage of the molecule through the nanopore. A nanopore can be asynthetic, man-made, biologically altered, wild-type biologicalnanopore, or a combination thereof.

A speed bump, as used herein, is an oligonucleotide molecule that formsa complex with a binding segment of a test DNA molecule. When the testDNA molecule goes through a nanopore under an electric potential, thecomplex formed between the speed bump and the binding segment stalls thetest DNA molecule in the nanopore for a dwelling time long enough forthe nanopore detector to obtain structure information of the test DNAmolecule. After the dwelling time, the complex dissociates and the testDNA molecule moves forward through the nanopore.

An identifier, as used herein, is a known structure in a test DNA thatcan be detected or identified by the method described herein. Examplesof identifiers include, without limitation, direction identifiers,reference signal identifiers, sample source identifiers, and sampleidentifiers. The identifiers may comprise one or more nucleotides orstructures that provide distinctive electrical signals that are easilyidentified. Examples of such nucleotides and structures include, withoutlimitation, isodG, isodC, methylated nucleotides, locked nucleic acids,universal nucleotides, and abasic nucleotides. In certain embodiments,an abasic nucleotide provides a stronger signal than a primarynucleotide. Thus, the electrical signal detected by a nanopore for asequence comprising both abasic nucleotides and primary nucleotides willprovide a signal more intense than the electrical signal obtained fromprimary nucleotide only sequences. For example, a 4˜5 base sequencecomprising about 25% abasic nucleotides may provide a signal more thantwice as strong as a 4˜5 base sequence comprising only primarynucleotides. The more abasic nucleotides the sequence have, the strongerelectrical signal the sequence. Thus, identifiers may provide electricalsignals of a desired intense (e.g. about twice, about 3, 4, 5, 6, 7, 8,9, or about 10 times stronger than that of primary oligonucleotideshaving the same length) by changing the amount of abasic nucleotides inthe identifier sequences.

Direction identifier, as used herein, is a known sequence positioned 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20bases from a bulky structure formed from a pre-bulky structure (theshaded section in the ss test DNA molecule, FIG. 9 ). When the bulkystructure is properly formed, it will stop the ss test DNA molecule fromflowing through a nanopore that the ss test DNA molecule is in. Thus,when the bulky structure is stopped inside the nanopore, a set ofelectrical signals will be obtained, which provide sequence informationof the sequence that is in front of the bulky structure and the firstbasepair of the bulky structure, in the flow direction of the ss testDNA molecule. When the sequence is known, such electrical signals can:

1) verify that the pre-bulky structure has properly formed into thebulky structure such that the bulky structure stops the ss test DNAmolecule from flowing through the nanopore;

2) indicate that the ss test DNA molecule has reached one end of thesingle strand section of the ss test DNA, and

3) serve as a reference or calibration read to base line otherelectrical signals obtained in the same nanopore.

In certain embodiments, the direction identifier comprises one or morenucleotides or structures that provide distinctive electrical signalsthat are easily identified. Examples of such nucleotides and structuresinclude, without limitation, isodG, isodC and abasic nucleotides.

Reference signal identifier, as used herein, is a known sequence in atest DNA, when detected or identified by the method described herein,serves as a reference or calibration read to base line other electricalsignals obtained in the same nanopore.

Sample source identifier, as used herein, is a known sequence in a testDNA, when detected or identified by the method described herein, is usedto identify the source that the sample DNA is from.

Sample identifier, as used herein, is a known sequence in a test DNA,when detected or identified by the method described herein, is used toidentify the individual sample DNA.

A known speed bump is a speed bump that specifically binds to a knownsequence in a ss test DNA. Because the binding segment on the ss testDNA (the known sequence) is known, the speed bump structure can also beknown (e.g. complementary to the known sequence on the ss test DNA).

A random speed bump pool, as used herein, comprises a collection ofspeed bumps that can bind to all sections of a test DNA molecule or afragment thereof. An example of random speed bump pool comprisesoligonucleotides having universal nucleobases which base-pair with allprimary nucleobases (A, C and G). Another example of random speed bumppool comprises oligonucleotides of a given length having all possiblecombinations of primary nucleobases. Another example of random speedbump pool comprises oligonucleotides of a given length having everypossible combination of primary nucleobases and universal nucleobases,Another example of random speed bump pool comprises speed bumps havinguniversal nucleobases at designated positions and all combinations ofprimary nucleobases at the other positions. Another example of randomspeed bumps is a combination of ss speed bumps, which form duplexsections with ss test DNA, and the duplex sections have about the samemelting temperatures. These ss speed bumps may have the same ordifferent lengths, and/or the same or different nucleotides.

“About,” as used herein, refers to +/−10% of the recited value.

The present invention is directed to a method for detecting and/oridentifying a sequence in a test DNA using a nanopore detector. The DNAsequence is trapped in the nanopore by one or two bulky structuresformed at the end(s) of the DNA sequence, so that the same test DNA canbe read multiple times by the same nanopore detector. Furthermore, eachbulky structure can be bound to the 5′ end or the 3′ end to thread thesample DNA into the pore in a known direction,

Known speed bumps are used to bind to known sequences in the test DNAfor the detection/identification of the known sequences. This method canbe used to detect whether the test DNA has correctly threaded into thenanopore, which sample source the test DNA is from, and individuallyidentify the test DNA trapped in the nanopore. Furthermore, the test DNAmay, further comprise a reference signal indicator to generateelectrical signals that can be used as reference of calibration forother electrical signals obtained form the same nanopore. Furthermore,multiple test DNAs can be analyzed by multiple nanopores at the sametime for efficient and simultaneous sequencings and characterizations.The multiple nanopores can be individually addressable and individuallyapplied desired electric potential, Thus, multiple test DNAs can beanalyzed simultaneously first to identify the DNAs having one or moredesired known sequences. The DNAs that do not have the desired knownsequence can be released without further characterizations. The DNAshaving the desired known sequences can be further characterized, andoptionally isolated and concentrated as described herein.

A random speed bump pool is used to bind to the test DNA or a fragmentthereof in a random fashion. Thus, each and every nucleotide of the testDNA or the fragment thereof will be stalled in the nanopore for a timelong enough to collect the nucleotide sequence information. Thenucleotide sequence of the test DNA or the fragment thereof can beidentified by taking all the sequence information obtained together. Thetest DNA may further comprise known structures such as directionidentifiers, reference signal identifiers, sample source identifiers,sample identifiers to provide information, e.g. formation of the bulkystructures, source of the test DNA, and identification of the test DNA.The test DNA may further comprise an isolation tag to isolate andconcentrate the test DNA. In certain embodiments, multiple test DNAs aredetected/identified by multiple nanopores (e.g. in a nanopore array).The method described herein can be applied to each test DNAdetected/identified. The nanopores can be individually addressed andcontrolled to selectively detect/identify/collect/concentrate testDNA(s) therein.

FIG. 1 shows an example of a nanopore detector having temperaturecontrol, which is prepared according to US Application Publication No.2011/0193570. The nanopore detector comprises electrodes (A), a treatedsemiconductor hydrophobic surface (B1) in which is embedded a conductiveelectrode (A) that is connected to electrical circuitry in asemiconductor substrate (B2), a layer of lipid material (C) created overan electrode (A) and a small portion of the hydrophobic surface closeby(B1), and a nanopore (D) inserted in the lipid material through aconductive salt solution (F). A sample detected goes through the pore(E) in the nanopore. The semiconductor chip sensor is placed in package(G) and this, in turn, is in the vicinity of a Peltier device ortemperature control element (H). Multiple nanopore detectors can becreated to form a nanopore array.

As illustrated in FIG. 2 , a single-stranded (ss) DNA molecule can gothrough a nanopore under an applied electric potential. A set ofelectrical signals corresponding to the brief blockages of ion flowthrough the nanopore by the ss test DNA molecule is detected as the sstest DNA molecule is threaded through the nanopore. In the absence ofspeed bumps or bulky structures, the ss test DNA molecule encounterslittle resistance and travels through the nanopore too quickly forelectrical signals to be reliably recorded for sequencing of the ss testDNA.

Bulky structures (BSs) have been used to stop the passage of a ss testDNA through a nanopore. FIG. 3 illustrates a trailing end BS used tostop the passage of a ss test DNA molecule through a nanopore. The BScan be a hairpin structure formed at one end of the ss test DNA bywrapping the trailing end of the ss test DNA upon itself. Typically, thess test DNA can be threaded through the nanopore under an appliedelectric potential until the bulky hairpin structure reaches theentrance of the nanopore. Since the hairpin structure is larger than thediameter of the nanopore, the ss test DNA is stalled in the nanoporelong enough to obtain a set of electrical signals of the ss test DNA.However, the electrical signals obtained reflects the structure of onlya portion of the DNA that is in front of the hairpin or in front of thespecific duplex region and therefore in or near the constriction area ofthe nanopore.

FIG. 4 illustrates a ss test DNA trapped in a nanopore by two bulkystructures. The nanopore detection is carried out at a workingtemperature that may be lower than room temperature so that one or moreshorter DNA duplex sections can be formed between speed bumps and the sstest DNA (speed bump-test DNA duplex segments). The speed bump-test DNAduplex segment stalls the ss test DNA for a sufficient dwelling time toobtain sequence information of the ss test DNA segment in front of thespeed bump-test DNA duplex segment and the first basepair of the speedbump-test DNA duplex segment in the flow direction of the ss test DNA.Then the speed bump-test DNA duplex segment dissociate and the ss testDNA moves forward through the nanopore until stalled by another speedbump-test DNA duplex segment or stopped by a bulky structure on one endof the ss test DNA. Once the ss test DNA reaches one end, the electricpotential can be optionally at a reduced value or a reversed polarity tomove the ss test DNA to a reversed direction and repeat the process asdesired.

When the ss test DNA has an unknown sequence e.g. sample DNA), a randomspeed bump pool can be constructed to bind to random sections of the sstest DNA. As every section of the ss test DNA can be bound by at leastone speed bump in the random speed bump pool, the binding patternsachieved by contacting a ss test DNA with a random speed bump pool eachtime can be random (FIG. 5 ). Thus, the segments whose sequenceinformation is obtained are also random for each run (FIG. 6 ). However,repeating the process as described supra allows each and everynucleotide of the unknown sequence to be identified by the nanoporedetector. Thus, the whole unknown sequence can be constructed byoverlapping the obtained sequence information of random sections of thess test DNA.

When the ss test DNA comprises one or more known sequences(identifiers), the method described herein can also be used to detectthe presence of one or more identifiers and/or to identify a sequence onthe ss test DNA that is in front of the identifier in the flow directionof the ss test DNA. The ss test DNA can have BS on only one end (FIG. 7) or both ends as described supra. The nanopore detector is operated ata working temperature lower than room temperature. A speed bump poolcomprises speed bumps that can bind specifically to the identifier (e.g.identifier 1, FIG. 7 ) to form a speed bump-identifier duplex segment isused. The speed bump-identifier duplex segment stalls the ss test DNAand a set of electrical signals are obtained. These signals can becharacterized to show presence of the identifier or to identify thesequence of the segment before the identifier in the flow direction ofthe ss test DNA. An example of such electrical signals is shown in FIG.8 .

Design and Construction of Test DNAs from a Sample DNA

In one embodiment, a sample DNA is linked with various functionalmoieties to facilitate nanopore sequencing and/or identifications.Examples of functional moieties include, without limitation, pre-bulkystructures and identifiers as described supra, and isolation tags tofacilitate isolation and enrichment of the sample DNA. The functionalmoieties optionally comprise one or more nucleotides.

As used herein, a nucleotide can be a primary nucleotide or a nucleotideanalog. A primary nucleotide is deoxyadenosine mono-phosphate (cAMP),deoxycytidine mono-phosphate (dCMP), deoxyguanosine mono-phosphate(dGMP) or deoxythymidine mono-phosphate (dTMP). A nucleotide analog isan analog or mimic of a primary nucleotide having modification on theprimary nucleobase (A, C, G and T), the deoxyribose structure, thephosphate group of the primary nucleotide, or any combination thereof.For example, a nucleotide analog can have a modified base, eithernaturally existing or man-made. Examples of modified bases include,without limitation, methylated nucleobases, modified purine bases (e.g.hypoxanthine, xanthine, 7-methylguanine, isodG), modified pyrimidinebases (e.g. 5,6-dihydrouracil and 5-methylcytosine, isodC), universalbases (e.g. 3-nitropyrrole and 5-nitroindole), non-binding base mimics(e.g. 4-methylbezimidazole and 2,4-diflurotoluene or benzene), and nobase (abasic nucleotide where the nucleotide analog does not have abase). Examples of nucleotide analogs having modified deoxyribose (e.g.dideoxynucleosides such as dideoxyguanosine, dideoxyadenosine,dideoxythymidine, and dideoxycytidine) and/or phosphate structure(together referred to as the backbone structure) includes, withoutlimitation, glycol nucleotides, morpholinos, and locked nucleotides.

The sample DNA may be a synthetic DNA or a DNA obtained from abiological sample. In one embodiment, the sample DNA has 1 to about100,000 bases, 1 to about 10,000 bases, 1 to about 1,000 bases, 1 toabout 500 bases, 1 to about 300 bases, 1 to about 200 bases, 1 to about100 bases, about 5 to about 100,000 bases, about 5 to about 10,000bases, about 5 to about 1,000 bases, about 5 to about 500 bases, about 5to about 300 bases, about 5 to about 200 bases, about 5 to about 100bases, about 10 to about 100,000 bases, about 10 to about 10,000 bases,about 10 to about 1,000 bases, about 10 to about 500 bases, about 10 toabout 300 bases, about 10 to about 200 bases, about 10 to about 100bases, about 20 to about 100,000 bases, about 20 to about 10,000 bases,about 20 to about 1,000 bases, about 20 to about 500 bases, about 20 toabout 300 bases, about 20 to about 200 bases, about 20 to about 100bases, about 30 to about 100,000 bases, about 30 to about 10,000 bases,about 30 to about 1,000 bases, about 30 to about 500 bases, about 30 toabout 300 bases, about 30 to about 200 bases, about 30 to about 100bases, about 50 to about 100,000 bases, about 50 to about 10,000 bases,about 50 to about 1,000 bases, about 50 to about 500 bases, about 50 toabout 300 bases, about 50 to about 200 bases, or about 50 to about 100bases.

Pre-Bulky Structures

In one embodiment, a ss test DNA comprises a first pre-bulky structure(PB1) on a first end that can form a first bulky structure (BS1) under afirst condition and a second pre-bulky structure (PB2) on a second endthat can form a second bulky structure (BS2) under a second condition.In certain embodiments, P131 comprises ss DNA segments that can form BS1under the first condition. A first condition can be a first temperatureT1, which can be about room temperature to 70° C., about 40° C. orhigher, about 30° C. or higher, about 25° C. or higher, about 20° C. orhigher, or about 15° C. or higher. In certain embodiments, the firstcondition can be T1 and the presence of a first ligand that can bind toPB1 to form BS1. Examples of the first ligand include, withoutlimitation, antisense oligonucleotide to PB1, other compounds tofacilitate formation of BS1 (e.g. compounds that can form a binding-pairwith a ligand, wherein the ligand is attached to PB1. Examples of suchbiding-pairs include, without limitation, antibody-antigen, andbiotin-streptavidin system), and combinations thereof through covalentand/or noncovalent interactions. Wherein BS1 is a DNA 2-D or 3-Dstructure (e.g. duplex, hairpin structure, multi-hairpin structure andmulti-arm structure), the melting temperature of BS1 (Tm1) is 15° C. orabove, about 20° C. or above, about 25° C. or above, about 30° C. orabove, about 35° C. or above, about 40° C. or above, or about 50° C. orabove.

PB2 forms BS2 under a second condition. In certain embodiments, PB2 is ass DNA segment that can form BS2 (e.g. DNA duplex, hairpin structure,multi-hairpin structure and multi-arm structure) under the secondcondition. A second condition can be a second temperature T2, it isabout −5 to about 50° C., about 40° C. or higher, about 30° C. orhigher, about 25° C. or higher, about 20° C. or higher, about 15° C. orhigher, about 10° C. or higher, or about 5° C. or higher. In certainembodiments, T2 is about at least 5° C. lower, preferably at least about10° C. lower or at least about 20° C. lower than T1. In certainembodiments, the second condition can be T2 and the presence of a secondligand that can bind to PB2 to form BS2. Examples of the second ligandinclude, without limitation, antisense oligonucleotide to PB2, othercompounds to facilitate formation of BS2, (e.g. compounds that can forma binding-pair with a ligand, wherein the ligand is attached to PB2.Examples of such biding-pairs include, without limitation,antibody-antigen, and biotin-streptavidin system), and combinationsthereof through covalent and/or noncovalent interactions, Wherein BS2 isa DNA 2-D or 3-D structure (e.g. duplex, hairpin structure,multi-hairpin structure and multi-arm structure), the incitingtemperature of BS2 (Tm2) is about 5˜about 10° C., about 10˜about 20° C.,about 20˜about 30° C., or about 20˜about 50° C.

In certain embodiments, PB1 and/or PB2 comprise(s) structures that arenon-binding to speed bumps in the speed bump pool. Examples of suchstructures include, without limitation, nucleotide analogs comprisingnon-binding bases such as IsodG, IsodC and abasic site.

Identifiers

In certain embodiments, a ss test DNA further comprise(s) functionalmoieties such as identifiers and isolation tags. In certain embodiments,when the ss test DNA is contacted with a random speed bump pool,identifier and isolation tags are constructed such that they will not bebound by the random speed bump pool. For example, an identifier segmentcan have isodG and isodC bases which preferably bind to each other. Ifspeed bumps of the random speed bump pool do not have isodG or isodCbase, speed bumps from the random speed bump pool will more preferablybind to sections of the ss test DNA that is outside of the identifiersegments. Thus, fewer electrical signals will be collected relating tothe sequence information of the identifier, which makes the collectedelectrical signals easier to characterize.

Examples of identifiers include, without limitation, directionidentifiers, reference signal identifiers, sample source identifiers andsample identifiers.

A ss test DNA may have only one bulky structure on one end (FIG. 7 ), ortwo bulky structures on both ends (FIG. 9 ). One direction identifierwill be positioned closely (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 bases) to each bulky structure inthe ss test DNA. The direction identifiers for different bulkystructures can be the same or different.

When the ss test DNA has two bulky structures and two directionidentifiers, other identifiers can be positioned between the twodirection identifiers. When the ss test DNA has only one bulky structureon one end and one direction identifier, other identifiers can bepositioned further away from the bulky structure compared to thedirection identifier.

Other identifiers include, without limitation, reference signalidentifier serves as a reference or calibration read to base line otherelectrical signals obtained in the same nanopore; sample sourceidentifiers used to identify the source of the sample DNA; and sampleidentifiers used to identify individual sample DNAs.

Because the structures of the identifiers are known, an identifier canbe detected and/or identified by contacting an identifier-specific speedbump with the ss test DNA. If the ss test DNA comprises the identifierof interest, an identifier-specific speed bump duplex section will beformed, which will stall the ss test DNA in the nanopore. A set ofelectrical signals will be obtained while the ss test DNA is stalled inthe nanopore, which can be used to indicate the formation of the speedbump-identifier duplex section (identifier, FIG. 10 ) and/or identifythe sequence that is in front of the identifier-speed bump duplex andthe first basepair of the identifier-speed bump duplex, in the flowdirection of the ss test DNA molecule (shaded section, FIG. 10 ). FIG.10 shows the situation in a ss test DNA having only one bulky structure.The same method can be used when the ss test DNA has bulky structures onboth ends.

In certain embodiments, the identifiers and/or identifier-specific speedbumps and/or the sequence in front of the identifier in the flowdirection of the ss test DNA molecule comprise one or more nucleotidesor structures that provide distinctive electrical signals that areeasily identified. Examples of such nucleotides and structures include,without limitation, nucleotides comprising isodG or isodC, abasicnucleotides, methylated nucleotides, etc.

Isolation Tags

An isolation tag is a structure that can form a binding-pair with aligand, wherein the ligand is further modified to facilitateconcentration or isolation thereof. Examples of such biding-pairsinclude, without limitation, antibody-antigen, and biotin-streptavidinsystem. Examples of further modifications to facilitate concentration orisolation include, without limitation, attachment of the ligand to amagnetic bead that can be easily concentrated and/or isolated.

In certain embodiments, more than one functional moieties may overlapwith each other or serve for more than one function.

An example of a ss test DNA comprising multiple functional moieties(segments A, B, C, D, F, G, H, and I, FIG. 11 ) and a sample DNA isshown in FIG. 11 .

Segment I may serve as a pre-bulky structure and forms a bulky structurewith a complementary strand thereof or by self-folding into a structure(e.g. DNA hairpin structures, multi-hairpin structures and multi-armstructures), and segment H or a fragment thereof can serve as adirection identifier. Alternatively, segment I can form a hairpin withsegment H under certain conditions. Thus, in this case, a pre-bulkystructure is segments I and H. Segment G or a fragment thereof can serveas a direction identifier.

Segment F, G, and H, or a fragment thereof can be a reference signalidentifier, a sample identifier, a sample source identifier.Alternatively, segments F, G and H together can be an identifier, or anyfragment thereof can also serve as an identifier described supra.Similar situations apply to segments A, B, C and D on the 3′ end. AnIsolation Tag can be placed on the 3′ end or on the 5′ end, and it canbe linked to the 3′ terminal or 5′ terminal nucleotide, or to anynucleotide on segments A, B, C, D, F, G, H and I, as long as it does notinterfere with the binding of speed bump to the ss test DNA, function ofnanopore, or formation of bulky structure.

Construction of ss Test DNA Comprising Sample DNA and One or MoreFunctional Moieties

A test DNA comprising sample DNA and one or more functional moieties isconstructed by ligating the sample DNA with other segments as desiredusing conventional organic and/or biological methods can be designed asdescribed supra

The ss test DNA shown in FIG. 11 can be formed by linking multiplefunctional moieties to a sample DNA using conventional ligation methods(e.g. formation of covalent bonds (e.g. ligase assisted ligation orother covalent bonds, wherein the ligation can be accomplished by pairedend sequencing chemistry, blunt-ended DNA ligation, and/or sticky-endligation) or non-covalent interactions).

In certain embodiments, sample DNA obtained is a double-stranded (ds)sample DNA. The ds sample DNA can be ligated with one or more dsfunctional moieties (e.g. ds PB1 (Segments I&H-Segments A′&B′, FIG. 12), ds PB2 (Segments B&A-Segments H′&I, FIG. 12 ), ds identifiers (e.g.(Segments G&F-Segments C′&D′, and Segments D&C-Segments F′&G′, FIG. 12), etc.) using conventional ligation methods (e.g. ligase assistedligation following blunt end, dangling end, and/or linker ligation;mate-paired and end-paired protocols.) (FIG. 12 ). The functionalmoieties can be ligated to the sample DNA all in one step, orsequentially, or all functional moieties on one end of the sample DNAare constructed together first and then ligated to the end of the sampleDNA. Examples of the conventional ligation methods includes, withoutlimitation, ligase assisted ligation following blunt end, dangling end,and/or linker ligation; paired end sequencing protocols; mate-paired andend-paired protocols. The obtained ds DNA is then denatured to providess test DNA using conventional methods (e.g. heated to denature the dsDNA).

In certain embodiments, the sample DNA obtained is a ds sample DNA, andis linked to one or more ds functional moieties (e.g. ds PB1, ds PB2, dsidentifiers etc.) via covalent bonds other than the phosphodiesterbonds. Examples of such linkage include, without limitation, the linkagein glycol nucleotides, morpholinos, and locked nucleotides.

In certain embodiments, the sample DNA obtained is a ss sample DNA, andits complementary strand can be created to anneal with the ss sample DNAto form a ds sample DNA using conventional methods, and then ligate toone or more ds functional moieties as described supra.

In certain embodiment, a ss sample DNA is linked to one or more ssfunctional moieties (e.g. ss PB1, ss PB2, ss identifiers etc.) usingligase assisted ligation. In certain embodiments, a ss sample DNA islinked to one or more ss functional moieties via covalent bonds otherthan the phosphodiester bonds. Examples of such linkage include, withoutlimitation, the linkage in glycol nucleotides, morpholinos, and lockednucleotides.

In certain embodiment, the sample DNA obtained is a ds sample DNA andcan be denatured to provide a ss sample DNA to be linked to one or moress functional moieties as described supra.

In certain embodiments, the functional moieties are linked by cleavablebonds such that one or more individual functional moieties can becleaved from the ss test DNA. In one embodiment, a bulky structure canbe removed from a ss test DNA by cleaving a functional moietiespositioned between the sample DNA and the bulky structure. Then, the sstest DNA can be released from the nanopore it is in by applying anelectric potential to move the ss test DNA through the nanopore in thedirection at which it is no longer stopped by the cleaved bulkystructure.

In certain embodiments, desired functional moieties are linked at adesired end (3′ or 5′) of the sample DNA, such that the test DNAobtained thereof can be threaded into the nanopore at a desireddirection (e.g. from 3′ end or from 5′ end).

Identification of a Sample DNA Using a Random Speed Bump Pool

One aspect of the invention relates to a method of identifying a sampleDNA sequence comprising:

-   -   (A1) providing a double-stranded (ds) sample DNA;    -   (A2) ligating a first pre-bulky (PB1) structure to a first end        of the ds sample DNA, and ligating a second pre-bulky (PB2)        structure to a second end of the ds sample DNA,    -   (A3) denaturing the ds sample DNA of A2 to a ss test DNA,    -   (B1) forming a first bulky structure (BS1) from PB1 on the first        end of the ss test DNA at a first temperature,    -   (B2) applying a first electric potential to flow the ss test DNA        through a nanopore,    -   (B3) forming a second bulky structure (BS2) from PB2 on the        second end of the ss test DNA at a second temperature,    -   (B4) optionally applying another electric potential to reverse        the flow of the ss test DNA until the ss test DNA is stopped by        BS2 before the constriction area of the nanopore,    -   (B5) contacting a random speed bump pool with the ss test DNA to        form a speed bump-ss test DNA complex having at least one speed        hump-ss test DNA duplex segment at a working temperature,    -   (B6) applying a third electric potential to flow the speed        bump-ss test DNA complex through the nanopore until a first        speed bump-ss test DNA duplex segment is stopped before the        constriction area of the nanopore,    -   (B7) obtaining a first set of electrical signals when the first        speed bump-ss test DNA duplex segment is stalled inside the        nanopore for a dwelling time, and characterizing the nucleotide        sequence that is in front of the first speed bump-ss test DNA        duplex segment and the first basepair of the first speed bump-ss        test DNA duplex segment, in the flow direction of the ss DNA,    -   (B8) dissociating the first speed bump-ss test DNA duplex        segment and continuing the flow of the ss DNA through the        nanopore, and    -   (B9) repeating steps (B4)˜(B8) until the ss test DNA is stopped        by BS1 or BS2,

In one embodiment, the ss DNA is a ss test DNA comprising a sample DNAas described supra. Speed bumps comprise one or more nucleotides asdefined supra.

A random speed bump pool comprises a collection of speed bumps of agiven length that can bind to all sections of the ss test DNA or afragment thereof (e.g. a sample DNA). Such a given length can be 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, preferred 10 or less, 8or less, 6 or less and 4 or less. In one embodiment, the random speedbump pool comprise speed bumps composed of one or more nucleotidesselected from the group consisting of universal nucleotides, lockednucleotides, primary nucleotides, modifications thereof, andcombinations thereof. Modifications of universal nucleotides, andprimary nucleotides include modifications at the nucleobase structures,the backbone structures (e.g. glycol nucleotides, morpholinos, andlocked nucleotides) and combinations thereof. In a preferred embodiment,the random speed bump pool comprises oligonucleotides having universalnucleobases which base-pair with all primary nucleobases (A, T, C andG). In another preferred embodiment, the random speed bump poolcomprises oligonucleotides having all possible combinations of primarynucleobases. In another preferred embodiment, the random speed bump poolcomprises oligonucleotides having all possible combinations of primarynucleobases and universal nucleobases. In another preferred embodiment,the random speed bump pool comprises oligonucleotides having universalnucleotides at designated positions and all combinations of primarynucleobases at the other positions. In another preferred embodiment, thebackbone structures of the speed bumps in the random speed bump pool aremodified (e.g. glycol nucleotides, morpholinos, and locked nucleotides)at designated position(s), random positions or combinations thereof.

The speed bumps comprise universal nucleobases at designated positionsand random primary nucleobases at other positions to lower the totalnumber of possible combinations of primary nucleobases. For example, fora random speed bump pool having 10-base oligonucleotides, the totalamount of combinations of the primary nucleobases is 4¹⁰=1,048,576.However, if 4 positions of the 10-base nucleotide are designated to haveuniversal nucleobases only, the total amount of combinations of theprimary nucleobases is 4⁶=4,096, which is significantly lower.

In certain embodiments, because the first base pair of the speedbump-test DNA duplex segment may be partially or completely in thenanopore and influence the electrical signals obtained, it is preferredto construct the speed bumps to have a universal nucleotide at the 5′and/or 3° end to normalize the contribution of the first base pair ofthe speed bump-test DNA duplex segment and makes the signals easier toanalyze.

In certain embodiment, the concentrations of one or more speed bumps ofa random speed bump pool may be further adjusted to as desired. Forexample, the concentrations may be about the same for each type of speedbump; and be adjusted such that sufficient ss speed bumps exist tocontact the ss test DNA. In one embodiment, because polyG strands bindstrongly to polyC strands, polyG and polyC speed bumps will have higherconcentrations than speed bumps having other sequences to providesufficient ss polyG and ss polyC to contact the ss test DNA. In anotherembodiment, the concentrations of speed bumps and/or nucleotides used tomake the speed bumps are adjusted such that each speed bump has aboutthe same affinity to form speed bump-test DNA complex, and no specificspeed bumps are significantly more favored than others. In certainembodiments, the concentrations of speed bumps and/or nucleotides usedto make the speed bumps are adjusted such that one or more specificspeed bumps are significantly more favored than others. For example, thespeed bump pool can be constructed to be substantially free of speedbumps that can bind to known segments in the ss test DNA. Therefore,more sequence information obtained will be about the unknown segmentsand not the known segments in the ss test DNA.

In certain embodiments, step (B5) forms a speed bump-test DNA complexhaving at least one speed bump-test DNA duplex segment, wherein thespeed bump forms a duplex with the ss test DNA segment that is up to 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 0.3, 14, 15, or 16 basepairs, and isthreaded in the nanopore at a first working condition. A workingcondition includes parameters such as a working temperature (Tw),exposure time, concentration of speed bump and ss test DNA, pH, saltconcentration, and other additives and concentration thereof that canaffect the formation of speed bump-test DNA complex. Tw is about −10 toabout 25° C., about −10 to about 20° C., about −10 to about 15° C.,about −10 to about 10° C., about −10 to about 5° C., about −10 to about0° C., about −10 to about −5° C., about −5 to about 25° C., about −5 toabout 20° C., about −5 to about 15° C., about −5 to about 10° C., about−5 to about 5° C., or about −5 to about 0° C., to allow association ofrelatively short speed bumps (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or 16 bases) to the ss test DNA. In one embodiment, Tw is aboutat least 10° C. lower, preferably at least about 20° C. lower than T2.In another embodiment, at Tw, at least about 50% of PB1 and PB2 are inthe forms of BS1 and BS2, respectively. In another embodiment, at Tw, atleast about 70% of PB1 and PB2 are in the forms of BS1 and BS2,respectively. In another embodiment, at Tw, at least about 90% of PB1and PB2 are in the forms of BS1 and BS2, respectively.

Exposure time of ss test DNA to speed bumps is about 1 ns or longer,about 10 ns or longer, about 1 μs or longer, about 10 μs or longer,about 1 ms or longer, about 10 ms or longer, about 1 s or longer, orabout 5 s or longer to allow sufficient speed bump-test DNA complex toform. Concentrations of the speed bumps are preferably about 100,000times, 10,000 times, 1,000 times, 300 times, about 200 times, about 100times, about 50 times, or about 20 times of the concentration of the sstest DNA, or the concentration of the speed bumps is about the same asthat of the ss test DNA. The concentrations of the speed bumps arepreferably about 1 nM about 100 mM, about 1 nM˜about 10 mM, about 1nM˜about 1 mM, about 10 nM˜about 100 mM, about 10 nM˜about 10 mM, about10 nM˜about 1 mM, about 1 mM˜about 10 mM, or about 10 mM˜100 mM. Theconcentration of ss test DNA is about 1 nM˜about 100 mM, about 1nM˜about 10 mM, about 1 nM˜about 1 mM, about 10 nM˜about 100 mM, about10 nM˜about 10 mM, or about 10 nM˜about 1 mM. pH is preferably about6˜about 8, or about 7. Salt (e.g. KCl, NaCl, phosphate) concentration isabout 1 mM to about 10 M, about 1 mM to about 1 M, about 10 mM to about10 M, about 10 mM to about 1 M, about 100 mM to about 10 M, or about 100mM to about 1 M. Other additives that may affect the formation of speedbump-test DNA complex include, without limitation, dextran sulfate andglycerol. Their concentrations may be adjusted to optimize formation ofspeed bump-test DNA complex.

A working condition further comprises an electric potential of about0-mV to about 320 mV at a desired polarity. The working condition can becontinuously adjusted through the process based on the characteristicsof the speed bump binding (e.g. length, nucleotide components, andbinding affinity), the nanopore characteristics and the ss test DNAproperty (e.g. GC content or secondary structure thereof), to optimizethe signal quality. Thus, the electric potential can continuously changefrom for example, −320 mV to +320 mV.

Steps (B4)˜(B9) are carried out at a first working condition asdescribed supra. In certain embodiments, the electric potential appliedto each step of steps (B4)˜(B9) may be the same or different orcontinuously changing. In certain embodiment, the electric potential forstep (B8) may be adjusted to facilitate the dissociation of the speedbump-test DNA duplex segment. In certain embodiment, the electricpotential for step (B8) may be applied to move the ss test DNA at areversed direction compared to the ss test DNA flow direction in step(B6) (forward direction) to move the speed bump-test DNA duplex segmentfrom the constriction area of the nanopore before applying anotherelectric potential to move the DNA at the forward direction todissociate the speed bump-test DNA duplex segment.

A dwelling time required for a nanopore detector to collect relevantsequence information relates to the nanopore detector and the workingcondition. In certain embodiments, the dwelling time is at least about10 μs, at least about 1 ms, at least about 10 ms, at least about 200 ms,at least about 500 ms, at least about 1 s, at least about 2 s, or atabout least 5 s. Generally, the longer the dwelling time is, the betterthe signal quality, and the more sequence information that can beobtained. In certain embodiments, the sequence of up to 5 bases isidentified when a speed bump-test DNA duplex segment is stalled in ananopore. In certain embodiments, the sequence of up to 3 bases isidentified when a speed bump-test DNA duplex segment is stalled in ananopore. In certain embodiments, the sequence of up to 2 bases isidentified when a speed bump-test DNA duplex segment is stalled in ananopore. In certain embodiments, the sequence of 1 bases is identifiedwhen a speed bump-test DNA duplex segment is stalled in a nanopore.

As shown in FIG. 4 , a ss test DNA comprising bulky structures formed onboth ends is locked in a nanopore (steps (B1)˜(B4)) and forms speedbump-test DNA complex with multiple speed bumps (step (B5)).

A set of electrical signals of the ss test DNA are obtained each timethe ss test DNA is stalled by a speed bump-test DNA duplex segment inthe nanopore for a dwelling time, and then the speed bump-test DNAduplex segment dissociates and the ss test DNA moves forward untilstalled by the next speed bump-test DNA duplex segment (FIG. 5 , the sstest DNA is illustrated to move from Cis side to Trans side). Thisstall-detect-disassociate-stall process is repeated until the ss testDNA is stopped by the bulky structure of one end. An example ofelectrical signals obtained is shown in FIG. 8 .

In certain embodiments, a random speed bump pool is present mainly onone side of the nanopore (e.g., Cis side as shown in FIG. 5 ), and themethod further comprising:

-   -   (B10) applying another electric potential to move the ss test        DNA at a reversed direction of the ss test DNA flow in step (B5)        until the ss test DNA is stopped by the other bulky structure        before the constriction area of the nanopore,    -   (B11) repeating steps (B4)˜(B10) at least 1 time, at least 5        times, at least 10 times, at least 15 time, at least 20 time, at        least 25 times, at least 30 times, at least 50 times, or at        least 100 times and    -   (B12) constructing the ss test DNA sequence by overlapping the        collected nucleotide sequence information.

Step (B10) are carried out under a working condition described supra.The electric potential applied can be at a reduced value or a reversepolarity compared to the electric potential applied in step (B4)˜(B9) toreverse the flow of the test DNA. The electric potential applied in eachstep can be the same or different or continuously changing.

In certain embodiments, a random speed bump pool is present in bothsides of the nanopore and speed bumps bind to the ss test DNA at thesegment exposed to the speed bump pool in both sides of the nanopore(Cis and Trans sides as shown in FIG. 13 ). The method of identifying anucleotide sequence of a sample DNA in a ss test DNA described hereinfurther comprising:

-   -   (1) repeating steps (B4)˜(B8) under a second working condition        until the ss test DNA is stopped by the other bulky structure        before the constriction area of the nanopore.    -   (2) repeating steps (B9) and (1) at least 1 time, at least 5        times, at least 10 times, at least 15 time, at least 20 time, at        least 25 times, at least 30 times, at least 50 times, or at        least 100 times; and    -   (3) constructing the nucleic acid sequence of the sample DNA by        overlapping the collected nucleotide sequence information.

The second working condition is a working condition as described supra.The second working condition can have the same or different parameterscompared to the first working condition. The electric potential appliedin step (1) is at a reduced value or a reverse polarity compared to theelectric potential applied in step (B9). The electric potential appliedin each step can be the same as applied in the earlier step, ordifferent compared to the earlier step, or continuously changing.

Because a random speed bump pool comprises speed bumps that can bind torandom sections of the ss test DNA, each time when the ss test DNA goesfrom one end stopped by BS1/BS2 to the other end according to theprocess described herein, speed bumps may bind to different combinationsof ss test DNA duplex segments (FIG. 5 ), and can provide sequenceinformation of different segments in the ss test DNA (FIG. 6 ). Thus,when step (B8) and/or step (B9) are/is repeated such that sequenceinformation of each and every nucleotide of the sample DNA in the sstest DNA has been obtained, the sample DNA can be constructed byoverlapping the collected nucleotide sequence information.

In certain embodiments, more than one speed bump is linked by anon-biding linker (e.g. abasic oligonucleotide) to form speed bump train(FIG. 14 ) such that the dissociation of each speed bump-test DNA duplexsegment will not cause the dissociation of the whole speed bump trainfrom the ss test DNA. In certain embodiments, the non-binding linker isdesigned to be spaced by about 1 base, about 2 bases, about 3 bases,about 4 bases or about 5 bases. Thus, the gap between known segmentsshown in FIG. 6 will be more likely to be the same as the length of thelinker (e.g. about 1 base, about 2 bases, about 3 bases, about 4 basesor about 5 bases). It will be easier to construct the nucleic acidsequence of the sample DNA in this case.

In certain embodiment, a method described herein wherein:

-   -   step (B2) further comprises:    -   (B2a) obtaining a set of electrical signals when the first bulky        structure is stalled inside the nanopore, and characterizing the        nucleotide sequence that is in front of the first bulky        structure and the first basepair of the first bulky structure,        in the flow direction of the ss test DNA, and step (B3) further        comprises:    -   (B3a) obtaining another set of electrical signals when the        second bulky structure is stalled inside the nanopore, and        characterizing the nucleotide sequence that is in front of the        second bulky structure and the first basepair of the second        bulky structure, in the flow direction of the ss test DNA.

In one embodiment, a method as described herein is carried out accordingto a flowchart shown in FIG. 15 . A ss test DNA comprising PB1, PB2,DI1, DI2 and a sample DNA has been constructed and placed on nanoporearray (Block 10, FIG. 15 ). Then BS 1 is formed from PB1 on one end ofthe ss test DNA at T1 (Block 20, FIG. 15 ). A first electric potentialis applied to thread the ss test DNA through a nanopore until the sstest DNA is stopped by BS1 wherein a set of electrical signalscharacterizing DI1 are collected (Block 30, FIG. 15 ). The temperatureis then lowered to T2 to form BS2 from PB2 (Block 40, FIG. 15 ). Asecond electric potential that is lower than the first electricpotential or opposite in polarity to the first electric potential isapplied until the ss test DNA is stopped by BS2 wherein a set ofelectrical signals characterizing DI2 are collected (Block 50, FIG. 15). The temperature is further lowered to Tw (Block 60, FIG. 15 ), thencontact a random speed bump pool with the ss test DNA under a firstworking condition as described supra to form randomly bound speedbump-test DNA complex (Block 70, FIG. 15 ). A third electric potentialis applied, moving the speed bump-test DNA complex through the nanoporeuntil the ss test. DNA is stalled by a first speed bump-test DNA duplexsegment. The ss test DNA is stalled for a dwelling time during which aset of electrical signals are obtained, which will be used tocharacterize the sequence in front of the first speed bump-test DNAduplex segment and the first base pair of the speed bump-test DNA duplexsegment in the flow direction of the ss test DNA. Then the first speedbump-test DNA duplex segment is dissociated and the ss test DNAcontinues through the nanopore until stopped by the next speed bump-testDNA duplex segment or BS1. A set of electrical signals designated to DI1are collected when the ss test DNA is stopped by BS1 in the nanopore(Block 80, FIG. 15 ). Then a fourth electric potential that is at areduced value or a reverse polarity to the third electric potential isapplied until the ss test DNA is stopped by BS2 wherein a set ofelectrical signals characterizing DI2 are collected (Block 90, FIG. 15). Then the steps in Blocks 70˜90 are repeated until sufficient sequenceinformation has been collected to characterize the sequence of thesample DNA.

Detection of an Identifier and Identification of an Identifier in a TestDNA Molecule

Another aspect of the invention relates to a method of obtainingsequence information of a ss test DNA molecule as described supra. Themethod comprises:

-   -   (B1) forming a first bulky structure on a first end of the test        DNA molecule,    -   (C1) contacting a pool of speed bumps (speed bump pool) with the        test DNA molecule to form a speed bump-test DNA molecule complex        having at least one speed bump-test DNA molecule segment,    -   (C2) applying an electric potential to flow the speed bump-test        DNA molecule complex through a nanopore until a first speed        bump-test DNA molecule segment is stalled before the        constriction area of the nanopore,    -   (C3) obtaining a first set of electrical signals when the first        speed bump-test DNA molecule segment is stalled inside the        nanopore for a dwelling time, in the flow direction of the test        DNA molecule,    -   (C4) dissociating the first speed bump-test DNA molecule segment        and continuing the flow of the molecule through the nanopore,        and    -   (C5) repeating steps (C1)˜(C4) until the test DNA molecule is        stopped by BS1.

In one embodiment, the test DNA molecule is a ss test DNA comprising oneor more nucleotides as described supra, and the speed bumps comprise oneor more nucleotides as described supra. The ss test DNA comprises PB1 asdescribed supra.

In certain embodiments, step (C1) forms a speed bump-test DNA complexhaving at least one speed bump-test DNA duplex segment, wherein thespeed bump forms a duplex with the test DNA duplex segment that is up to1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 basepairs.

Steps (C1)˜(C5) are carried out at a working condition as describedsupra.

A dwelling time required for a nanopore detector to collect relevantsequence information is the same as described supra.

In certain embodiment, the method described herein is used to detect anidentifier exists in the ss test DNA. An identifier can serve as e.g.direction identifier (e.g. verifying the formation of BS1 and showingthe ss test DNA has reached to the end having BS1), reference signalidentifier (a reference or calibration read to base line otherelectrical signals obtained in the same nanopore), sample sourceidentifier (identifying the source of the test DNA), or sampleidentifier for the test DNA (identifying the test DNA). In certainembodiments, a speed bump pool comprises a first speed bump (FIG. 7 )which can bind to a first identifier (identifier 1 in FIG. 7 ), and issubstantially free of other speed bumps that can bind to the ss test DNA(preferably less than 10%, more preferably less than 5%, and mostpreferably less than 1%). When a ss test DNA comprising identifier 1contacts the first speed bump, a first speed bump-identifier 1 duplexsegment is formed to form a first speed bump-test DNA complex. In thepresence of an appropriate electrical field, the first speed bump-testDNA complex goes through a nanopore until stalled by the first speedbump-identifier 1 duplex segment. The nanopore detector obtains a firstset of electrical signals. Then the first speed bump-test DNA complexdissociates and the ss test DNA goes through the nanopore until stoppedby BS1 at the first end (i.e. in step (C4), the ss test DNA flow throughthe nanopore smoothly until stopped by BS1 without being stalled againin the nanopore). The nanopore detector will obtain another set ofelectrical signals when the ss test DNA is stopped by the BS1 structure.Thus, compared to a ss test DNA that does not comprise identifier 1sequence, the ss test DNA that comprises identifier 1 sequence providestwo sets of electrical signals showing that it is stalled twice in thenanopore, while the ss test DNA that does not comprise identifier 1sequence provides one set of electrical signals showing it is stalledonce in the nanopore (by BS1).

In another embodiment, the ss test DNA and/or the speed bumps can beconstructed such that the first set of electrical signal obtained instep (C3) is distinctive from a set of electrical signals obtained whena primary nucleotide sequence is detected by the nanopore. For example,the known identifier sequence can comprise one or more nucleotideanalogs having isodG and/or IsodC. In front of this identifier sequenceis a known reading sequence that would be in the constriction zone of apore if a speed bump was hybridized to the identifier sequence andstopped in the pore. The reading sequence could comprise IsodC, IsodGand/or abasic positions that do not bind to natural nucleotides.Additionally, both the identifier sequence and the specific antisensespeed bump sequence to the identifier would contain appropriate IsodGand IsodC so that only the specific speed bump to the identifier wouldhybridize to that location. Natural nucleotide speed bumps would notinterfere or bind to the IsodG, IsodC-containing identifier sequence andnatural nucleotide speed bumps would not interfere with the readingsequence. The resulting identification of the strand in the pore wouldoccur independent of the presence of other natural or man-madenucleotide speed bumps. In this case, the speed bump pool does not haveto be substantially free of other speed bumps that can form complex withthe ss test DNA. When another speed bump binds to a segment of the sstest DNA other than identifier 1 segment, the first set of electricalsignal obtained while the first speed bump-test DNA duplex segment isstalled in the nanopore is distinctive from the other set of electricalsignal obtained while the other speed bump-test DNA duplex segments arestalled in the nanopore. Thus, the presence of other speed bumps thatcan form complex with the ss test DNA does not interfere with thedetection of the distinctive signals generated from binding of the firstspeed bump with identifier 1 of the ss test DNA. The ss test DNA and/orthe speed bumps can be further constructed such that no other speedbumps binds to the identifier 1 segment as described supra. Thus, otherspeed bumps that do not comprise isodG or isodC bases will not bind tothe identifier 1 segment.

In another embodiment, the ss test DNA comprises more than oneidentifier, and the ss test DNA and/or the speed bumps (SBN, N=1, 2, . .. ) that bind to the identifier segments (identifier N) respectively aredesigned such that when each SBN-identifier N duplex segment is stalledin the nanopore, the set of electrical signal obtained from the nanoporeis distinctive from a primary nucleotide sequence and from when otherSBN-identifier N duplex segment is stalled in the nanopore. The speedbump pool comprises the speed bumps specific for the identifier(s) thatis(are) to be detected, and optionally include speed bumps for otheridentifiers and/or other speed bumps that can bind to the ss test DNA.

In another embodiment, the identifier that binds to theidentifier-specific speed bump and the sequence in front of theidentifier in the flow direction of the ss test DNA are both known.Thus, the set of electrical signals obtained in step C3 can also be usedto identify the sequence in front of the identifier in the flowdirection of the ss test DNA, which can in turn be used to identify ofthe identifier.

In another embodiment, the method further comprises applying a firstelectric potential to flow the ss test DNA through a nanopore, andforming a second bulky structure (BS2) on a second end of the ss testDNA under a second condition as described supra. In one embodiment, thetemperature of the first condition (T1) is higher than the temperatureof the second condition (T2), which is higher than the workingtemperature Tw. In a preferred embodiment, the temperature of the firstcondition (T1) is at least 10° C. higher or at least 20° C. higher thanthe temperature of the second condition (T2), which is at least about 1°C. higher, at least about 5° C. higher, at least about 10° C. higher, atleast about 15° C. higher, at least about 20° C. higher, or at leastabout 25° C. higher than the working temperature Tw.

In certain embodiments, the identifier sequence that is adjacent to thesample DNA is contacted with a first known speed bump (e.g. 2, 3, 4, 5,6, 7, 8, 9, or 10 bases long), and the sample DNA sequence that is infront of the identifier-speed bump duplex in the first flow direction ofthe ss test DNA molecule can be identified. Extending the sequence ofthe known speed bump in the flow direction of the ss test DNA allowsidentification of longer sequences in the sample DNA. This methodcomprises the following steps:

-   -   (E1) contacting a first known speed bump with the test DNA        molecule to form a first known speed bump-test DNA molecule        complex having a first known, speed bump-test DNA molecule        segment,    -   (E2) applying an electric potential to flow the first known        speed bump-test DNA molecule complex through a nanopore until        the first known speed bump-test DNA molecule segment is stalled        before the constriction area of the nanopore,    -   (E3) obtaining a first set of electrical signals when the first        known speed bump-test DNA molecule segment is stalled inside the        nanopore for a dwelling time, in the flow direction of the test        DNA molecule.    -   (E4) dissociating the first known speed bump-test DNA molecule        segment and continuing the flow of the molecule through the        nanopore,    -   (E5) removing the first known speed bumps from the nanopore        detector system and reversing the flow of the test DNA until        stopped by the bulky structure at the end, and    -   (E6) repeating steps (E1)˜(E5) with another known speed bump        having a sequence of the first known speed bump plus a known        number of bases longer in the flow direction of the test DNA        molecule of step (E3), wherein:        -   E-a) the known number is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,        -   E-b) the known number of bases can be universal bases or            bases that are complementary to the bases at the            corresponding positions of the sample DNA, and        -   E-c) the condition of step (E4) may be adjusted, e.g.            raising the working temperature and/or increasing the            electric potential value applied in step (E4) to dissociate            the speed bump-test DNA molecule segment successfully

Although the method described supra can only identify a sequence of thesample DNA not longer than 15 bases. Such knowledge may facilitateidentification/sequencing of the rest of the unknown sequence of thesample DNA using the method described supra (e.g. using a random speedbump pool). Furthermore, the same process can be used to identify asequence of the sample DNA from another end. Thus, up to 30 bases of anunknown sample DNA can be identified, which will provide a goodreference in further identification/sequencing of the Whole sequence ofthe sample DNA.

Isolation of Sample DNA

Another aspect of the invention relates to a method of isolating asample DNA comprising:

-   -   preparing a ss test DNA using steps (A1)˜(A3) as described supra    -   (D1) converting one of the two bulky structure such that the        corresponding end of the ss test DNA can go through the nanopore        without being stopped, and    -   (D2) applying an electric potential to release the ss target        DNA.

In certain embodiments, the ss test DNA further comprises an isolationtag as described supra, and the method further comprises step (D3) afterStep (D2):

-   -   (D3) attaching the isolation tag to a ligand.

In certain embodiments, wherein the ligand is further attached to amagnetic bead, step (D3) further comprising:

-   -   (D3-1) removing the conducting salt solution comprising the        released ss test DNA,    -   (D3-2) attaching the isolation tag to a ligand by mixing the        released ss test DNA with the ligand attached to a magnetic        bead, and    -   (D3-3) isolating the released ss test DNA using conventional        isolation methods.

In certain embodiments, the method further comprising step (D4) afterstep (D3):

-   -   (D4) removing the isolated ss test DNA from the bead using        conventional methods (e.g. using a basic solution), and    -   (D5) cleaving PB1 and PB2 from the ss test DNA to generate the        ss sample DNA.

In certain embodiments, step (D5) further comprising cleaving PB1 andPB2 using endonucleases. In certain embodiments, step (D5) furthercomprising cleaving PB1 and PB2 at a cleavable site.

In one embodiment, step (D1) further comprises:

-   -   (D1-1) changing the temperature of the nanopore to about or        higher than the second temperature and lower than the first        temperature to convert BS2 to a non-bulky structure.        Sequencing, Identification, Concentration and Isolation of        Sample DNAs Using Multiple Nanopore Detectors.

Another aspect of the invention relates to a method of sequencing,identifying, concentrating and isolating of sample DNAs using multiplenanopore detectors. The same method as described herein regarding singlenanopore detector can be used to multiple nanopore detectors.

In one embodiment, the multiple nanopores are individually addressable,wherein the electric potential of each nanopore can be individuallycontrolled. The temperature of the nanopore may also be controlled.Thus, the ss test DNA molecules detected in a nanopore can beindividually released by carrying out steps (D1)˜(D3) on selectednanopores.

For example, in an array of nanopore (numbered as N1, N2, . . . N10),each nanopore has a ss test DNA trapped according to the methoddescribed herein, and the individual DNA is numbered DNA1, DNA2, . . .DNA10 in the corresponding nanopores N1, N2, . . . N10. If only DNA1 andDNA 3 are desired to be collected, nanopores N1, N2, . . . N10 can beindividually controlled such that only DNA1 and DNA3 are collected (e.g.by applying an electric potential to move DNA1 and DNA3 from nanoporesN1 and N3 respectively). In one embodiment, BS2s of DNA1, DNA2, . . .DNA10 are converted to a structure that can go through the nanopores(e.g. PB2s at a temperature about or higher than the second temperaturewhile lower than the first temperature, or cleaved to leave ss structurethat can go through the nanopores, respectively). The electric potentialof the nanopores N2, N4˜N10 are individually controlled such that DNA2,DNA4˜DNA10 are released from the nanopores respectively, while DNA1 andDNA3 are still trapped in nanopores N1 and N3, respectively. Thennanopores N1 and N3 are individually controlled to release DNA1 andDNA3, respectively to be collected, concentrated and/or isolated.

The invention is illustrated further by the following examples that arenot be construed as limiting the invention in scope to the specificprocedures or products described therein.

EXAMPLES Example 1. PB2 Structure (I)

A ss test DNA having a BS2 on one end was captured in a nanopore at atemperature lower than T2 and released at a temperature higher than T2(FIG. 16 ).

The BS2 (BS2-1) was a DNA 5-base duplex hairpin structure formed from aPB2 having a sequence of 5′-CCCCC CCCCC TTATA CCCCT ATAA-3′ (SEQ ID NO.1, PB2-1). BS2-1 had melting temperature of about 15° C., and a ΔG ofabout −0.96 kcal/mol at 5° C. according to the simulation using UNAFOLDprogram. This moderately low ΔG indicated that BS2-1 had a relativelylow binding energy.

In FIG. 16 , the solid line showed the change in temperature from 2° C.to 14° C. The dots represented individual DNA captures, meaning thatPB2-1 formed BS2-1 at the corresponding temperature and was captured inthe nanopore. The captures were present when the temperature was aboutor lower than T2 (about 5° C.), indicating that BS2-1 was formed fromPB2-1 and the DNA was stalled in, the nanopore. The capture of the DNAdisappeared when the temperature increased to about 5˜10° C. over T2,indicating that BS2-1 melted and was no longer stalled in the nanopore.

Thus, PB2-1 formed BS2-1 which stopped the ss DNA in the pore at atemperatures about 10° C. lower than its melting temperature. This maybe due to the relatively low ΔG BS2-1 had. Thus, the DNA duplexstructure in BS2-1 was relatively easy to dissociate in the nanopore.Thus, a BS2 having a higher ΔG may be more difficult to destruct and mayprovide longer dwelling time at the nanopore at a temperature closer tothe melting temperature of the BS2.

Example 2. PB1 and PB2 Structure (II)

A PB1 forms a BS1 at a first temperature (T1) that is higher than thesecond temperature (T2) at which a BS2 is formed from a PB2. T2 ishigher than a working temperature (Tw). In this example, Tw is belowroom temperature. Thus, PB1 is designed to have a relative long DNAduplex segment (either in a DNA duplex with an anti-sense DNA segment,or in a hairpin structure) such that the desired melting temperature ofthe relative long DNA duplex segment is achieved.

PB2 is designed to have a lower melting temperature and a high bindingenergy (ΔG=about −1˜−5 kcal/mol, about −4˜−6 kcal/mol, about −4˜−5kcal/mol, about −4.5 kcal/mol, or about −4.0 kcal/mol at the workingcondition). A molly bolt or branched molecule has been designed toprovide a BS2 having low T2 while not easily dissociated at the workingcondition.

An example of PB1 has a sequence of 15 bases and a 4 base A loop;5′-CGTCT AGCGT TGCCG AAAAC GGCAA CGCTA GACG-3′ (SEQ ID NO. 2, PB1-1).This sequence has a melt temperature of 91.4° C. in 1 M KCl, and 1 μMsequence concentration according to the simulation using UNAFOLDprogram.

An example of PB2 has a sequence of 5′-GACCC TGCCC CCAGC TTTCC CCAAACGTCA AAAAA-3′ (SEQ ID NO. 3, PB2-2) and the formed BS2-2 is a 3 stem, 3duplex, 2 loop molecule as shown below according to the simulation usingUNAFOLD program:

The following characteristics of the BS2-2 were provided using UNAFOLDprogram:

ΔG=−4.5140 kcal/mol at 5° C. (100% folded),

ΔH=−67.90 kcal/mol,

ΔS=−227.9 cal/(K·mol) and

Tm=24.8083° C.

A calculated melting curve of BS2-1 was obtained using nearest neighborbasis and shown in FIG. 17 . This melting curve illustrates that atabove 30° C. about 90% of the structures are linear (PB2-2) and at below20° C. about 90% of the structures form BS2-2. Such a steep meltingcurve shows well controlled bulky structure formation of BS2-2, which ishighly desired. The ΔG of BS2-2 at 5° C. is −4.5 kcal/mol, whichindicates a stronger binding affinity than the 5 base hairpin moleculeBS2-1 in Example 1.

Example 3. Stalling DNA by 4-Base Duplex Segments

This example illustrates a 4-baser duplex segment stalled the ss testDNA in a nanopore for a dwelling time sufficient to obtain desiredsequence information.

The test DNAs were the following:

A test DNA was formed by self-hybridization of DNA-1: 5′-CCCCC CCCCCGCGC-3′ (SEQ ID NO. 4). DNA-1 was dissolved in biology grade water,heated to 90 CC and then left to cool to room temperature forself-hybridization. A DNA-1 molecule hybridize with another DNA-1molecule to form a self-hybridized DNA-1 structure having a 4-base GCGCduplex segment at the 3′ ends and two overhanging ss 10-C tails at the5′ ends thereof. At the working condition, the self-hybridized DNA-1structure entered a nanopore with one of the two overhanging ss 10-Ctails, stalled in the nanopore by the 4-base duplex segment at the 3′end for a dwelling time, and then when the 4-base duplex segmentdissociated, the self-hybridized DNA-1 structure was converted to two ssDNA-1 molecules which went through the nanopore like ss test DNAs. Thus,when flowing through a nanopore, the self-hybridized DNA−1 structuresimulated a ss test DNA having a 4-base duplex segment formed by a speedbump and the ss test DNA.

Another test DNA, self-hybridized DNA-2 structure, was formed byself-hybridization of DNA-2: 5′-TTTTT TTTTT GCGC-3′ (SEQ ID NO. 5) usingthe same process described supra regarding the formation of theself-hybridized DNA-1. The self-hybridized DNA-2 structure had a 4-baseGCGC duplex at the 3′ ends and two overhanging ss 10-T tails at the 5′ends.

Another test DNA was streptavidin-DNA-3 complex formed by incubation ofDNA-3: 5′-TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT-biotin-3′ (SEQID NO. 6) and streptavidin at a condition described below. When flowingthrough a nanopore under a electric potential, streptavidin-DNA-3complex stalled in the nanopore until the electric potential waschanged/reversed. Thus, streptavidin-DNA-3 complex served as a positivecontrol showing that the nanopore detector system was working properly.The dwell time of this molecule was long, represented by the points atthe far right side of the x-axis (time) in FIG. 18 .

The working condition was 20 mM HEPES buffer and 1 M KCl at 0° C. Theelectric potential applied was about 128 mV.

The nanopores were created from 10 ng/mL alpha hemolysin deposited ontothe surface of a bilayer at a final concentration of 0.2 ng/mL and withthe application of electrical stimulus as described in US ApplicationPublication No. 2011/0193570. The bilayers were created with thepainting method from 10 mg/mL of DPhPC in Decane across the essentiallyplanar AgCl electrode on a Teflon surface as described in US ApplicationPublication 2011/0193570.

Self-hybridized DNA-1 (2 μM self-hybridized DNA-2 (2 μM), DNA-3 (2 μM),and streptavidin (1 μM) were incubated with multiple nanoporesconstructed as described supra for about 2 h at the working conditiondescribed supra in this example. An electric potential of about 128 mVwas applied to the nanopore and electrical signals were collected andshown in FIG. 18 . FIG. 18 showed that the 4-base duplex segments wereable to stall DNA-1 and DNA-2 in the nanopore for a dwelling time ofabout 100 ms to 200 ms. These data showed that speed bumps as short as 4bases worked to stall a ss test DNA long enough to obtain relevantsequence information.

Example 4. Stalling DNA by 6-Base Random Speed Bump Pool

This example illustrates a 6-base random speed bump pool successfullybound to, stalled in a nanopore detector and dissociated from a testDNA.

In this example, the ss test DNA was ss female genomic DNA. The randomspeed bump pool comprised hexamer DNA oligonucleotides having allcombinations of the primary DNA nucleotides, which was purchased fromInvitrogen.

The working condition was 20 mM HEPES buffer and 1 M KCl at 0° C. Theelectric potential applied was about 128 mV.

The nanopores were created from 10 ng/mL alpha hemolysin deposited ontothe surface of a bilayer at a final concentration of 0.2 ng/mL and withthe application of electrical stimulus as described in US ApplicationPublication. No. 2011/0193570. The bilayers were created with thepainting method from 10 mg/mL, of DPhPC in Decane across the essentiallyplanar Aga electrode on a Teflon surface as described in US ApplicationPublication. No. 2011/0193570.

The ss test DNA (1 μM) was incubated with the 6-base random speed bumppool (100 μM) were incubated with multiple nanopores constructed asdescribed supra for about 2 h at the working condition described suprain this example. An electric potential of about 128 mV was applied tothe nanopore and electrical signals were collected. The signals showedthat the 6-base random speed bump pool was able to bind to the ss testDNA, stall the ss test DNA in the nanopore long enough to obtainrelevant sequence information, and dissociate from the ss test DNA asdescribed herein.

The invention claimed is:
 1. A method for sequencing a moleculecomprising a plurality of nucleotides or nucleotide analogs, the methodcomprising: applying a first electric potential having a first polarityacross an array of nanopore detectors, each nanopore detector comprisinga pair of electrodes, a membrane disposed between the pair ofelectrodes, and a nanopore disposed in the membrane; threading aplurality of molecules through the array of nanopore detectors in afirst direction based on the first polarity of the applied firstelectric potential; measuring a first electrical signal in each nanoporedetector as the molecule is threaded through the nanopore in the firstdirection; applying a second electric potential having a second polarityacross the array of nanopore detectors, wherein the second polarity isopposite the first polarity; threading the plurality of moleculesthrough the array of nanopore detectors in a second direction based onthe second polarity of the applied second electrical potential, whereinthe second direction is opposite the first direction; applying a thirdelectrical potential across the array of nanopore detectors, wherein thethird electrical potential has a polarity that is the same as thepolarity of the first electrical potential; threading the plurality ofmolecules through the array of nanopore detectors in the first directionbased on the polarity of the third electrical potential; measuring asecond electrical signal in each nanopore detector as the molecule isthreaded through the nanopore in the first direction as a result of theapplication of the third electrical potential; determining a sequencefor each molecule based on the first electrical signal and the secondelectrical signal; and applying a fourth electrical potential across thearray of nanopore detectors, wherein the fourth electrical potential hasa polarity that is the same as the polarity of the first electricalpotential, wherein the fourth electrical potential has a magnitude thatis greater than the first electrical potential that is configured toadvance the molecule past a speed bump that is slowing or stalling thethreading of the molecule through the nanopore.
 2. The method of claim1, wherein each molecule has a first end comprising a first bulkystructure.
 3. The method of claim 2, wherein the first end is thetrailing end of the molecule and the first bulky structure is configuredto prevent the molecule from passing completely through the nanoporewhen the molecule is threaded through the nanopore in the firstdirection.
 4. The method of claim 2, wherein each molecule has a secondend comprising a second bulky structure.
 5. The method of claim 4,wherein the second end is the leading end of the molecule and the secondbulky structure is configured to prevent the molecule from passingcompletely through the nanopore when the molecule is threaded throughthe nanopore in the second direction.
 6. The method of claim 2, whereinthe first end is the leading end of the molecule and the second firststructure is configured to prevent the molecule from passing completelythrough the nanopore when the molecule is threaded through the nanoporein the second direction.
 7. The method of claim 1, wherein each moleculecomprises a plurality of speed bumps located along a length of themolecule, wherein the plurality of speed bumps are configured to slow orstall the threading of the molecule through the nanopore.
 8. The methodof claim 1, wherein the first electrical potential, the secondelectrical potential, and the third electrical potential have amagnitude that is less than the magnitude of the fourth electricalpotential.
 9. The method of claim 1, wherein the first electricalpotential, the second electrical potential, and the third electricalpotential have a magnitude that is too low to reliably advance themolecule past a speed bump that is slowing or stalling the threading ofthe molecule through the nanopore.